Differentiation of Haemonchus placei from Haemonchus contortus by PCR and by morphometrics of adult parasites and third stage larvae / Identificação de Haemonchus placei e Haemonchus contortus por PCR e por análise morfológica de parasitas adultos e larvas infectantes

Molecular and morphological methods were evaluated to distinguish between Haemonchus contortus and Haemonchus placei species. A total of 141 H. contortus and 89 H. placei male adult specimens collected from artificially infected lambs were identified individually by PCR analysis, using a species-specific primer pair. These PCR results were used as gold standard for Haemonchus spp. identification. Haemonchus placei presented higher mean spicule and barb lengths than H. contortus (P<0.05). However, some measurements overlapped. For this reason, a discriminate function did not allow the correct identification of 13 H. contortus and one H. placei specimen. The sheath tail length of the third stage larvae (L3), which comprises the distance between the tip of the larval tail and the end of the sheath tail, were measured. Only three of the 485 H. placei larvae (0.619%) had a sheath tail shorter than 85 µm, while only four of the 500 H. contortus larvae (0.8%) presented a sheath tail longer than 85 µm. The results indicated that 6.09% of the male adult specimens would be misclassified based on the discriminate function, while only 0.71% of infective larvae would be misclassified. Therefore, identification of L3 can be used as the first method to indicate the presence of H. placei and/or H. contortus in a population of domestic ruminants.

Métodos moleculares e morfológicos foram avaliados para a identificação de Haemonchus contortus e Haemonchus placei. No total, 141 H. contortus e 89 H. placei machos adultos, obtidos de cordeiros artificialmente infectados, foram identificados individualmente por PCR com o emprego de um par de “primers” espécie-específico. Esses resultados da análise por PCR foram considerados como padrão para a identificação das espécies de Haemonchus. Haemonchus placei apresentou valores médios de espículos e ganchos superiores aos de H. contortus (P<0,05). Entretanto, houve sobreposição de alguns valores. Por essa razão, a função discriminante não permitiu a identificação correta de 13 exemplares de H. contortus e de um, de H. placei. Foi medida a cauda da bainha de larvas infectantes (L3), que compreende a distância entre a ponta da cauda da larva e a ponta da cauda da bainha. Apenas três das 485 L3 de H. placei (0,619%) apresentaram a cauda da bainha com medida inferior a 85 µm e somente em quatro das 500 L3 de H. contortus (0,8%) essa medida foi superior a 85 µm. Os resultados demonstraram que 6,09% dos machos adultos seriam identificados erroneamente com base na função discriminante, enquanto a identificação incorreta de L3 seria de apenas 0,71%. Portanto, a identificação de L3 pode ser utilizada como método inicial para indicar a presença de H. placei e/ou H. contortus em uma população de ruminantes domésticos.

Catalytical properties of N-glycosylated Gluconacetobacter diazotrophicus levansucrase produced in yeast

The influence of N-glycosylation on the kinetic and catalytical properties of a bacterial fructosyltransferase (LsdA) produced in Pichia pastoris was studied. The glycosylated enzyme behaved similarly to non-glycosylated LsdA when substrate specificity, fructo-oligosaccharide (FOS) production, sucrose hydrolysis or levan formation reactions were carried out under different experimental conditions. The kinetic parameters for native or yeast-expressed LsdA determined at 60ºC, condition for the highest hydrolytic activity, followed a conventional Michaelis-Menten kinetics. Synthase activity of this levansucrase increased in water-restricted environments by addition of salt or organic solvent to the reaction mixtures.

Detección de meticilino-resistencia en Staphylococcus aureus: Comparación de métodos convencionales y aglutinación con mrsa-Screen latex / Methicillin resistance detection in Staphylococcus aureus: Comparison between conventional methods and Mrsa-Screen latex agglutination technique

Staphylococcus aureus meticilino-resistente (MRSA) es un patógeno que ha emergido en las últimas cuatro décadas causando tanto infecciones nosocomiales como de la comunidad. La rápida y precisa detección de MRSA es relevante para guiar una apropiada terapia antibiótica y evitar la diseminación nosocomial de MRSA.En este trabajo se evaluó la eficiencia de métodos convencionales para la detección de meticilino-resistencia como difusión por discos, CIM en medio sólido, screening de oxacilina, y el nuevo test de aglutinación MRSA-Screen latex sobre 100 aislamientos de S. aureus, 79 mecA positivos y 21 mecA negativos. El test de aglutinación MRSA-Screen latex (Denka Seiken, Niigata, Japón) detecta la presencia de la PLP-2a, producto del gen mecA en cepas de S. aureus. La detección del gen mecA por PCR se utilizó como gold standard para comparar los resultados de los diferentes métodos. La sensibilidad y especificidad fueron 97 y 100 % para el método de difusión, 97 y 95 % para la CIM en medio sólido, 100 y 100 % para el screening de oxacilina y 100 y 100 % para MRSA-Screen latex. Todos los métodos presentaron alta sensibilidad y especificidad, pero el “MRSA-Screen latex” mostró la ventaja de poder brindar un resultado confiable, equivalente a la PCR, en sólo 15 minutos.

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen that has emerged over the last four decades, causing both nosocomial and community-acquired infections. Rapid and accurate detection of methicillin resistance in S. aureus is important for the use of appropriate antimicrobial therapy and for the control of nosocomial spread of MRSA strains. We evaluated the efficiency of conventional methods for detection of methicillin resistance such as the disk diffusion, agar dilution, oxacillin agar screen test, and the latex agglutination test MRSA-Screen latex, in 100 isolates of S. aureus, 79 mecA positive and 21 mecA negative. The MRSA-Screen latex (Denka Seiken, Niigata, Japón), is a latex agglutination method that detects the presence of PLP-2a, product of mecA gene in S. aureus. The PCR of the mecA gene was used as the “gold standard” for the evaluation of the different methods tested. The percentages of sensitivity and specificity were as follows: disk difusión 97 and 100 %, agar dilution 97 and 95 %, oxacillin agar screen test 100 and 100 %, and MRSA-Screen latex, 100 and 100 %. All methods presented high sensitivity and specificity, but MRSA-Screen latex had the advantage of giving a reliable result, equivalent to PCR, in only 15 minutes.

Synthesis of fructans by fructosyltransferase from the tuberous roots of Viguiera discolor (Asteraceae)

Sucrose:sucrose fructosyltransferase (SST) and fructan:fructan fructosyl-transferase (FFT) activities from crude extracts of tuberous roots of Viguiera discolor growing in a preserved area of cerrado were analyzed in 1995-1996. SST activity was characterized by the synthesis of 1-kestose from sucrose and FFT activity by the production of nystose from 1-kestose. The highest fructan-synthesizing activity was observed during early dormancy (autumn), when both (SST and FFT) activities were high. The increase in synthetic activity seemed to start during the fruiting phase in the summer, when SST activity was higher than in spring. During winter and at the beginning of sprouting, both activities declined. The in vitro synthesis of high molecular mass fructans from sucrose by enzymatic preparations from tuberous roots collected in summer showed that long incubations of up to 288 h produced consistently longer polymers which resembled those found in vivo with respect to chromatographic profiles.